Microbubble aeration is supposed to be able to provide potential advantage for aerobic biological wastewater treatment due to enhancement of oxygen mass transfer. On the other hand, microbubble and its generation methods might affect mixed liquor properties of activated sludge. Then SPG membrane microbubble generation system was used to investigate variation of mixed liquor properties of activated sludge in microbubble aeration. The results indicated that sludge floatation happened in microbubble aeration due to attachment of microbubbles to sludge flocs, resulting in a decrease in mixed liquor suspended solid (MLSS) concentration and poor sludge settleability. The strong shear stress caused by liquid circulation pump during microbubble generation led to sludge broken, resulting in decreased sludge floc size and sludge organics release, such as extracellular polymers (EPS). The organics release from broken sludge flocs was the main reason for increased both supernatant organic content (especially organic colloids) and consequent supernatant turbidity. The re-flocculation ability of broken sludge flocs also depended on sludge EPS release. In addition, the viscosity of mixed liquor increased along with sludge broken and increased supernatant organic content but the surface tension of mixed liquor remained constant. These results displayed the possible problems to apply microbubble aeration in aerobic wastewater treatment processes based on activated sludge.